In 1980, the World Health Organization estimated that in developing countries food poisoning from infection with Salmonella bacteria (salmonellosis) contributed to more than 1 billion cases of acute diarrhea in children under the age of five years (Kvenberg and Archer, Food Technol. 40:77-98, 1987), and at least 5 million deaths (this reference, and all other references cited herein, is hereby expressly incorporated herein by reference in its entirety). Since the mid-1980s, the worldwide incidence of salmonellosis has increased steadily. S. enteritidis, in particular, has been implicated in the sharp increase in food borne infection since 1983. Indeed, the current frequency of S. enteritidis infections constitute a worldwide pandemic (Rodrique et al., Epidemiol. Infect. 105:21-27, 1990).
The severity of the disease is greatest in infants, the elderly, the infirm and in other persons with inadequate or impaired immune systems, including the malnourished. In third world countries where malnutrition is more commonly a complicating factor, mortality rates due to S. enteritidis infection as high as 28% have been reported. In both the clinical and industrial settings, the situation is also complicated by the fact that many people are asymptomatic carders. Salmonella spp., including S. enteritidis, often possess several plasmid encoded antibiotic resistance genes that complicate the treatment of human infections.
In the industrialized world, it is the contamination of food products by Salmonella bacteria that is most directly threatening to human health. Hence, it is not surprising that the increase in salmonellosis in first world countries parallels the centralization of food production and processing despite continued improvements in epidemiological and microbiological methods.
The significance of the problem is reflected in one aspect in the poultry-related industries. For example, in the U.S. alone hatcheries produce approximately 100 million broiler chicks per week and chicken egg production in the U.S. has reached 5 billion annually. A large proportion of S. enteritidis infections have been associated with the contamination of the contents of whole shell eggs resulting from vertical transmission of this pathogen from breeder stocks due to transovarian infection. This is significant since common procedures designed to decontaminate the external shell surface are not effective. The problem presented by S. enteritidis is exacerbated by the fact that infection in the adult laying hens may be asymptomatic. Typically, S. enteritidis infection of laying birds does not have a significant adverse effect on fertility, hatchability or egg production. Similarly, broiler chickens may be asymptomatic throughout their lifetime, although losses of about 20% do occur in infected flocks due to death in chicks, retardation of growth and rejection of contaminated birds at slaughtering. Contaminated poultry feed may be a major source of infection, but stress to poultry due to handling, transportation and overcrowding add to the problem by increasing the shedding of Salmonella from infected chickens. The end result is that the majority of modern processing plants, which process about 10,000 birds per hour, are contaminated and Salmonella are typically isolated from 40% to 70% of turkey or chicken carcasses sampled in the U.S. and Canada.
The overall economic costs of the rising incidence of food borne infections have been significant. The U.S. General Accounting Office has recently estimated the cost of S. enteritidis food poisoning in the U.S. between 1985 to 1990 at $118 million in lost productivity, medical and hospital costs resulting from about 9,500 illnesses. The U.S. Center for Disease Control receives more than 40,000 case reports annually but attributes greater than 2 million cases and roughly 2,000 deaths per year in the United States to salmonellosis (Cohen and Tauxe, Science 234:964-969, 1986). The economic cost related to treatment of salmonellosis in the U.S. was estimated to be $50 million in 1986. About 8 million cases involve physician consultation and an estimated 250,000 cases require hospitalization. Non-hospitalized cases are thought to have accounted for about $680 million in medical costs and minimally $2 billion in lost productivity. Others estimate the total costs of salmonellosis in the U.S. arising from medical treatment and lost productivity to be as high as $23 billion per year (Kvenberg and Archer, supra).
The losses absorbed by the food industry from liability and product loss are undoubtedly passed on to the consumer. Thus, there is a need for an effective risk-management program to monitor the different phases of poultry production including breeding, raising, slaughtering, packing and further processing, distribution and preparation, and consumption. The development of strategies for creating Salmonella-free feed, the control of Salmonella in breeder flocks, hatcheries, and product operations will include development of more effective diagnostics. Accordingly, there is a general need for a technology which could be applied to the inexpensive, rapid detection of all Salmonella food borne pathogens.
One type of assay for Salmonella comprises the standardized culture tests for Salmonella in the food industry. These culture assays are recognized by different names in different countries but they share the same basic approach. In the United States, the procedures are known as the "Bacteriological Analytical Manual" (BAM), published in 1984 by the Association of Official Analytical Chemists (AOAC, Arlington, Va.). In Canada, the procedures are known as the "Official Canadian Wet Culture Method" (WCM); the protocol most often used to test food samples is MFD-20.
Within the standardized tests, Samples are incubated at 30.degree. C. to 37.degree. C. for 18 to 24 hours in a rich, non-selective medium to promote recovery of the cells and allow them to begin to replicate to the levels detectable by current technologies. There will likely be an excess of other microorganisms in the sample, some of which may be from the closely related family Enterobacteriaceae. Therefore, a selective growth step is conducted to enrich for Salmonella bacteria, for example, by inoculating a small sample of the pre-enrichment culture into a selenite-cysteine broth, tetrathionite broth, or Rappaport-Vassiliadis broth for 18 to 24 hours, typically at an elevated temperature such as 43.degree. C. The cells are then plated on a selective medium, such as brilliant green agar or xylose-lysine-deoxycholate agar, and incubated overnight. Presumptive colonies are then transferred to various biochemical or metabolic test media for confirmation. Pure cultures of Salmonella are then grown overnight on agar slopes for serotyping. In total, three to four days are required to obtain presumptive positive results, and a five to seven day wait can be necessary before final confirmation and identification of the Salmonella.
An alternative test to assay for the presence of Salmonella is based on nucleic acid probes. One such test uses probes constructed from a part of the fimA gene of Salmonella typhimurium, and is preferentially based on two particular sequences (Madonna and Woods, EP Publication No. 383,509, Orthodiagnostic Systems, Raritan, N.J.). Briefly, a nucleic acid molecule of a known sequence is introduced to a sample under conditions suitable for hybridization of the nucleic acid molecule to its target nucleic acid sequence in the DNA or KNA of Salmonella. Alternative hybridization-based assays include the colormetric Gene-Trak.RTM. Salmonella Assay (Gene-Trak Systems, Framingham, Mass.), Fitts et al., Appl. Environ. Microbiol. 46:1146-1151, 1983.
Another alternative test is a fluorescent antibody assay (FA test Thomason, J. Food Protection 44:381-384, 1981), which includes the Salmonella Flouro-Kit (Incstar, Stillwater, Minn.). Such a test uses polyvalent antisera prepared against Salmonella flagella (anti-H) and lipopolysaccharide (LPS) O-chain (anti-O). The assay can also use purified polyclonal IgG antibodies. However, an FA test is laborious, has a high level of false-positive results, yields only presumptive positive samples, and requires visual determinations to be made by highly skilled personnel using expensive equipment.
Another test is an enzyme immunoassay (EIA). In general, as with the FA test described above, antibodies to flagella or lipopolysaccharide form the basis of most EIAs. EIAs can use either polyclonal or monoclonal antibodies. However, as with the FA test, false-positive results are a significant problem. Further, these assays can take an extensive testing period, and some diagnostic tests using monoclonal antibodies to Salmonella flagellin have reported significant problems with false-negative results. Examples of such EIAs include the TECRA Salmonella immunocapture ELISA manufactured by Bioenterprises Ply. Ltd., Roseville, NSW, Australia, and the Dynatech Laboratories, Inc. (Chantilly, Va.) Salmonella MICROELISA.RTM.-92 and MICROELISA.RTM.-32 Detection kit.
Still another type of test is an agglutination assay (Benge, Eur. J. Clin. Microbiol. Infect. Dis. 20 8:294-298, 1989), such as the Wellcollex-Colour Salmonella assay (Wellcome; Bouret and Jeanjean, J. Clinical Microbiology 30:2180-2186, 1992), which is based on anti-Salmonella antibodies conjugated to latex beads. This form of test is relatively simple, but requires at least two to three days to provide results from food or environmental samples, and has a relatively low level of sensitivity.
A further type of test is the selective motility assay, in which a sample potentially containing Salmonella is introduced into a pre-enrichment or selective growth medium in the first chamber of a double-chambered device. (Humbert et al., Letters in Applied Microbiology 10:245-249, 1990) The motile Salmonella then favorably grow and move across the pre-enrichment growth medium, entering the second chamber, which contains a semi-solid medium having a sample of antisera at the far end. As the motile Salmonella replicate and migrate into and across the second chamber, the antisera diffuses toward the oncoming bacteria, forming an immunoprecipitate line at the point where the bacteria contact the antisera.
Yet another test is a bacteriophage assay, such as the Vitek System's Salmonella test (McDonnell-Douglas Health Systems Co.). This assay uses bacteriophage that specifically recognize receptors on Salmonella. An enzyme is conjugated to the bacteriophage and is used for detection purposes. This test requires a minimum of 48 hours and is subject to false positive and false negative results.
Yet another test is an enzyme-linked amperometric immunosensor, a type of biosensor format (Brooks et al., Journal of Applied Bacteriology 73: 189-196, 1992).
Accordingly, the present invention discloses compositions and methods suitable for the diagnosis of Salmonella in a sample, including isolated nucleic acid molecules, isolated proteins, probes and primers.